CN115575646B - Application of metabolic marker group in preparation of kit for predicting epileptic seizure - Google Patents

Abstract

The invention relates to the field of epilepsy, in particular to application of a metabolic marker group in preparation of a kit for predicting epileptic seizures. The present invention provides the use of a metabolic marker set comprising L-threonine, homovanillic acid and glycine for the manufacture of a kit for assessing the risk of seizures in a subject who has been diagnosed with seizures and has a predisposition for seizures, said kit comprising a detection reagent for detecting the content of said metabolic marker set in a urine sample of said subject. The invention also provides the metabolic marker group.

Description

Application of metabolic marker group in preparation of kit for predicting epileptic seizure

Technical Field

The invention relates to the field of epilepsy, in particular to application of a metabolic marker group in preparation of a kit for predicting epileptic seizures.

Background

Epilepsy is one of the most common chronic neurological disorders, with over 7000 tens of thousands of patients worldwide. Seizures are caused by abnormal, self-sustaining discharges that occur suddenly in the brain network, usually lasting less than a few minutes. Symptoms of seizures can affect any part of the body, often appearing as sudden recurrence of sensory disturbance, loss of consciousness, or convulsions.

Since epilepsy is often abrupt and epilepsy itself lacks predictability, about 12 tens of thousands of people worldwide lose their lives each year. Accidental seizures are often accompanied by accidental death such as drowning, car accident injury, sudden epileptic death, etc., and thus may lead to accidents, injuries, embarrassments and expensive first-aid costs. It is clear that when seizures occur can place significant restrictions on family, society, education and occupational activities, and it can be seen that the social consequences of seizures are generally more significant than the effects of seizures themselves. In addition to the severe injury that falls and other accidents at the seizure may cause, the social dirty name associated with seizures and their unpredictability may lead to severe self-disclaimer, irritability, and anxiety in epileptic patients. Such anxiety may further lead to an increased incidence of seizures, and increased seizures may still further increase chronic anxiety.

In summary, seizures are difficult to predict and very dangerous. The epileptic seizure is predicted and early-warned in advance, so that the life quality of epileptic patients can be improved and enough time is provided for taking action in advance. The prior art generally uses electroencephalography (EEG) to analyze whether an epileptic patient is in seizure or not, and even predict seizure. However, this prediction is based on EEG data acquired from long-term monitoring of the brain signals of epileptic patients to predict an impending (e.g., within 1 hour) seizure. Thus, such predictions require a long period of time to continuously collect EEG data from epileptics and do not allow the epileptic sufficient time to take action to control the seizure (e.g., taking preventive medications) and to schedule a proper life plan in advance.

Disclosure of Invention

In one aspect, the invention provides the use of a metabolic marker set comprising L-threonine, homovanillic acid and glycine in the manufacture of a kit for assessing the risk of seizures in a subject diagnosed with seizures and having a predisposition for seizures, the kit comprising a detection reagent for detecting the content of the metabolic marker set in a urine sample of the subject.

In some embodiments, the subject is at least 18 years old.

In some embodiments, the subject is assessed as having a seizure risk for a predicted period when the content is higher than a reference content to which it is referenced.

In some embodiments, the predicted period is 1-3 days from the day of the urine sample collection.

In some embodiments, the L-threonine and the glycine are used to assess the seizure risk in a female subject; when the content of both the L-threonine and the glycine is higher than the reference content to which it is referenced, the female subject is assessed as strongly positive for seizures; when the content of the L-threonine or the glycine is higher than the reference content to which it refers, the female subject is assessed as seizure positive; the female subject is assessed as seizure-negative when neither the content of the L-threonine nor the glycine is higher than the reference content to which it is referenced.

In some embodiments, the L-threonine and the homovanillic acid are used to assess the seizure risk in a male subject; when the levels of both the L-threonine and the Gao Xiangcao acid are higher than the reference levels to which they refer, the male subject is assessed as strongly positive for seizures; when the content of the L-threonine or the homovanillic acid is higher than the reference content to which it refers, the male subject is assessed as seizure positive; the male subject is assessed as seizure-negative when neither the content of the L-threonine nor the homovanillic acid is higher than the reference content to which it is referenced.

In some embodiments, the subject is a drug refractory epileptic patient.

In some embodiments, the seizure type of the subject is of focal origin.

In some embodiments, the subject has an epileptic seizure at least once a month.

In some embodiments, the means of detection is selected from one or more of chromatography, mass spectrometry, a combination of chromatography and mass spectrometry, immunochemistry, fluorescence analysis, and radiochemical analysis.

In another aspect, the invention also provides a metabolic marker panel for assessing seizure risk in a subject diagnosed with seizures and having a seizure propensity, wherein the metabolic marker panel comprises L-threonine, homovanillic acid and glycine.

Compared with the prior art, the invention has the beneficial effects that:

prior art predictions of seizures typically rely on EEG data acquired by long-term monitoring of brain signals of epileptic patients. In some cases, the continuous EEG data that needs to be analyzed may be up to 24 hours, and may be read out of different results due to different analysis means (e.g., manual analysis and EEG-based machine learning model analysis). This not only takes a lot of time for the epileptic, the interpretation is subjective, but it does not allow the epileptic enough time to take actions to prevent and control seizures and to schedule a proper plan.

The invention is based on noninvasive, convenient and mass-available urine samples, and the risk of epileptic seizures within 1-3 days (from the date of urine sample collection) can be predicted by detecting the content change of specific metabolic marker groups (L-threonine, homovanillic acid and glycine) only by collecting urine of epileptic patients.

Compared with the prior art, the metabolic marker group provided by the invention provides a relatively longer response and preparation time for epileptics to take preventive measures or intervention measures in advance, for example, taking appropriate measures such as increasing the dosage in advance, preventing the epileptic seizure by means of vagal nerve stimulation or transcranial magnetic stimulation and the like, avoiding the causes of the epileptic seizure (such as staying up night and drinking alcohol) and the like. Therefore, the metabolic marker group provided by the invention can avoid other risks accompanied by sudden onset of epilepsy without early warning to a certain extent.

In addition, the technical scheme of the invention is based on the collection and detection of urine samples, so that the standardization of detection is relatively easier to realize, the detection cost is saved to a certain extent, and meanwhile, the early warning effect can be still realized. Of course, the metabolic marker set of the invention is also suitable for being matched with other detection modes to further predict epileptic seizure conditions.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the invention and that other drawings may be derived from them without inventive faculty.

FIG. 1 is a graph showing the results of a cluster analysis (analysis by Fuzzy C-Means) algorithm, hereinafter referred to as "FCM") of male patients according to a third embodiment of the present invention;

FIG. 2 is a graph showing the results of a cluster analysis (analysis by FCM) of male patients according to a third embodiment of the present invention;

FIG. 3 is a graph showing the results of L-threonine levels in male patients according to example III of the present invention;

FIG. 4 is a graph showing the results of the level of homovanillic acid in male patients of example III of the invention;

FIG. 5 is a graph showing the results of a female patient cluster analysis (analysis by FCM) according to a third embodiment of the present invention;

FIG. 6 is a graph showing the results of a female patient cluster analysis (analysis by FCM) according to a third embodiment of the present invention;

FIG. 7 is a graph showing the results of L-threonine levels in female patients according to example III of the present invention;

FIG. 8 is a graph showing the results of glycine levels in female patients according to example III of the present invention;

FIG. 9 is a summary of baseline information for an incorporated drug refractory epileptic patient according to an embodiment of the present invention;

FIG. 10 is a summary of elevated metabolites 1-3 days prior to seizure in male and female patients in accordance with example III of the present invention;

FIG. 11 is a summary of L-threonine and homovanillic acid levels before seizure in male patients in example IV of the invention;

FIG. 12 is a summary of L-threonine and glycine levels before seizure in female patients in example IV of the present invention;

fig. 13 is a schematic diagram of seizure risk assessment according to a fourth embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Herein, "and/or" includes any and all combinations of one or more of the associated listed items.

Herein, "plurality" means two or more, i.e., it includes two, three, four, five, etc.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

As used in this specification, the term "about" is typically expressed as +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, and even more typically +/-0.5% of the value.

In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, the description of ranges 1-6 should be considered as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within such ranges, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.

As used herein, the term "sample" or "biological sample" or "specimen" refers to biological material isolated from a subject. The sample may comprise any biological material suitable for detecting a desired biomarker, and may comprise cellular material and/or non-cellular material from a subject. The sample may be isolated from any suitable biological tissue or fluid, for example, blood, plasma, serum, skin, epidermal tissue, adipose tissue, liver tissue, urine, or cell samples. In some preferred embodiments, the sample is urine. The sample may be pretreated prior to actual testing by methods including filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like.

As used herein, the term "epileptic" refers to a clinical manifestation in which an individual has more than two non-evoked seizures. Exemplary seizures include extremely brief loss of consciousness or muscle reflex and severe and persistent twitches. The frequency of seizures varies from epileptic patient to epileptic patient, e.g., with lower frequency for example, fewer seizures per year; however, some patients have a high frequency of episodes that occur multiple times per day. In this context, exemplary seizure types include seizures of global and focal origin, exemplary classes of seizures include temporal lobe seizures, frontal lobe seizures, parietal lobe seizures, occipital lobe seizures, epileptic encephalopathy, and the like. The terms "seizure" and "epilepsy" are used interchangeably herein unless otherwise indicated.

The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal from which a sample is taken, unless otherwise indicated. A subject, individual, or patient may have, be at risk of, or be suspected of having a predisposition to a seizure or seizure symptomatic condition. In some embodiments, a typical subject includes a person susceptible to, suffering from, or having suffered from one or more seizures. In some preferred embodiments, the subject is an adult (i.e., the subject is 18 years old and older).

As used herein, the term "predisposition, e.g.," predisposition to have a seizure "refers to a reasonable medical probability of an event (e.g., occurrence or recurrence of a seizure). The term "predisposition" also includes the frequency with which such events may occur before, after or during continued treatment.

"content" or "level" of one or more biomarkers (simply "markers") refers to the absolute (quantitative) or relative (qualitative) amount or concentration of the biomarker in a sample.

As used herein, the term "metabolite" refers to any chemical or biochemical product of a metabolic process, typically in the form of a small molecule. Exemplary small molecules include sugars, fatty acids, amino acids, nucleotides, intermediates formed during cell processing, and other small molecules present within the cell.

Biological samples suitable for detecting markers include biological materials isolated from subjects. In some embodiments, the sample may be a blood, plasma, serum, skin, epidermal tissue, adipose tissue, liver tissue, urine, or cell sample. In some preferred embodiments, the sample is urine.

Any suitable method may be used to analyze a biological sample to determine the level of one or more markers in the sample. Exemplary suitable methods include immunochemical techniques such as chromatography (e.g., liquid Chromatography (LC), high Performance Liquid Chromatography (HPLC), gas Chromatography (GC)), mass spectrometry (e.g., mass Spectrometry (MS), tandem mass spectrometry (MS/MS 2)), combined methods (gas chromatography/mass spectrometry (GC-MS), liquid chromatography/mass spectrometry (LC-MS), ultra-high performance liquid chromatography/tandem mass spectrometry (UHLC/MS 2), and gas-chromatography/tandem mass spectrometry (GC/MS 2)), enzyme-linked immunosorbent assays (ELISA), and combinations thereof, refractive index spectroscopy (RI), ultraviolet spectroscopy (UV), fluorescent analysis, radiochemical analysis, near infrared spectroscopy (near IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS). In addition, in some embodiments, the content of one or more markers may also be measured by means of indirect measurement, for example measuring the content of a compound (or compounds) related to the content of the marker to be measured.

After determining the content of one or more markers in a biological sample obtained from a subject, the content is compared to a reference content to which it is referenced (e.g., a reference content derived from itself and/or a selected reference content (e.g., a content of a reference sample selected according to age, sex, type of epilepsy, etc.). If the levels of all markers in the subject's metabolic marker panel (in female subjects, "all" means L-threonine and glycine; in male subjects, "all" means L-threonine and homovanillic acid) are not significantly increased (i.e., are not higher) than the reference levels to which they refer (e.g., are the same as the reference levels, are substantially the same as the reference levels, are not statistically different from the reference levels, are within a predetermined range of the reference levels, etc.), then the subject is assessed as seizure-negative, i.e., the subject has a lower probability of seizures (can be considered as not seizures) within 1-3 days of the day that the biological sample was collected. The subject is assessed as seizure positive by an increased level of one or more markers in the metabolic marker set compared to its reference level, i.e., the subject has a higher probability of seizures within 1-3 days of the day that the biological sample was collected. In particular, if the content of all the markers of the metabolic marker set is increased compared to the reference content to which it is referenced, then the subject is assessed as strongly positive for seizures, i.e. the subject has a very high probability of seizures within 1-3 days of the day on which the biological sample was collected.

The "reference content" of a marker may be an absolute or relative amount or concentration of the marker, the presence or absence of the marker, a range of amounts or concentrations of the marker, the lowest and/or highest amounts or concentrations of the marker, an average amount or concentration of the marker, and/or a median amount or concentration of the marker. Suitable reference levels of the markers can be determined by measuring the desired marker levels of the same subject multiple times; it may also be determined by measuring the levels of markers required for a particular subject (e.g., subjects of the same gender and same age) as a reference level for a particular population. The reference content and the marker content of the subject may vary depending on the particular technique (e.g., LC-MS, GC-MS) used to measure the marker in the biological sample.

The marker content of a subject can be compared to a reference content to which it is referenced using a variety of means, such as simple comparison, statistical analysis (e.g., t-test, wilcoxon rank sum test (Wilcoxon rank sum test)). In some embodiments, the above comparison may be accomplished by a manual and/or automated system. In some embodiments, a subject's content of one or more markers that is 5%, 10%, 15%, 20% or more higher than the reference content to which it is referenced may be considered an increase in the content of one or more markers in the subject.

In some embodiments, for subjects with seizure frequency of at least 1 time per month on average, the reference content of the reference can be determined by regular collection (e.g., 1 time per day) over a period of time (e.g., 2 weeks). If the subject had seizure epilepsy during the acquisition, the content 1 week prior to its seizure can be considered as the reference content to which it was referenced.

Example 1

Subjects studied in accordance with the invention: the invention is incorporated into epileptics who visit the center of epilepsy at the Huaxi hospital of university of Sichuan, 4 th year 2021 to 8 th year 2022 (which are independently diagnosed as epileptics by two medical practitioners). Inclusion criteria for epileptic patients were: (1) Seizure frequency of approximately 6 months is 1 or more times per month on average; (2) age 18-50 years. The exclusion criteria were: (1) Patients with kidney disease, taking medications that clearly affect urinary protein changes or kidney function; (2) Patients with routine urine and abnormal liver and kidney function detection results; (3) Patients suffering from other neurological diseases such as cerebral apoplexy, brain tumor, alzheimer's disease, parkinson's disease, etc.; (4) patients with other chronic diseases; (5) gestational or lactating patients; (6) Patients who were unable to voluntarily or for other reasons, were unable to cooperate with the study of the present invention.

Baseline information of subjects studied in accordance with the invention: baseline information acquisition is accomplished face-to-face with the patient by the physician, including demographic information, epileptic-related medical history information, test results, and medical history, past history. Demographic information includes: gender, age, cultural degree, BMI. The epileptic related medical history information includes: the onset age of epilepsy, the course of the disease, the type of seizure, the duration of seizure, the severity score of seizure, whether it is drug refractory epilepsy, etc. The examination results include: skull MRI, skull PET-MRI, skull CT, common scalp electroencephalogram, video electroencephalogram, and the like. The medication history includes: the dosage, the type of the medicine, the dosage and the like. The past history includes: a history of birth hypoxia, a history of premature birth (gestational age <37 weeks), a history of febrile convulsion, a history of pre-onset encephalitis meningitis, a history of pre-onset cranium trauma, and a family history of epilepsy.

Considering that the seizures of most epileptic patients can be controlled after drug treatment, the invention selects drug refractory epileptic patients for key study, and the main baseline information is shown in fig. 9.

Urine sample collection and diary recording: urine collection is done autonomously by the subject at home. Each subject was continuously collected for 2 months, daily morning on-bed fasting first urination and first urination within 1h after each seizure. During collection, the middle-stage urine is collected by a disposable urine cup and poured into a 15mL centrifuge tube, and the tube is filled to 12-13mL, and 2 tubes are collected each time. After collection, the label with the serial number is attached to the centrifuge tube, and the date of the day and the time of collecting urine are written on the tube cover. If the urine is in a plurality of attacks within one day, urine after the attack is collected for 2 times at most and is separated by more than 1 h. Starting from the first day of urine collection, the subject recorded a diary daily in an electronic diary card. The recording content includes: (1) date when urine collection began; (2) day morning urine collection date and time; (3) Daily seizure condition and acquisition date and acquisition time of post-seizure urine; (4) Daily diet, exercise, sleep conditions and menstrual flow in women.

Urine transport and storage: after each collection, the subject immediately placed the centrifuge tube with the urine and labeled into a-20 ℃ refrigerator. The subjects sent urine every 2 weeks to the university of Sichuan Huaxi hospital with dry ice. The urine of the subject is received by a special person, is arranged and registered, and is finally stored in a refrigerator at the temperature of-80 ℃ in a biological sample library of Huaxi hospital of Sichuan university for standby.

Example two

Pretreatment of urine: first, urine samples were incubated with urease at a concentration of 100U to break down excess urea (considering high abundance urea as the primary chromatographic disturbance). After 50. Mu.L of the incubated sample was transferred to a centrifuge tube (the sample was run all the way on ice), 250. Mu.L of labelled methanol was added thereto, and the sample was vortexed at 1550rpm for 3min at 4 ℃. Next, the vortexed sample was stored in a refrigerator at-20℃for 20min, then ice-bathed for 15min, and finally centrifuged at 13300rpm for 15min at 4 ℃. 150. Mu.L of supernatant was taken into a fresh EP tube and concentrated in vacuo at 30℃for 2 hours. The dried extract was subjected to oximation treatment (2 hours at 60 ℃) using methoxyamine hydrochloride. Derivatization was then performed using MSTFA (N-methyl-N- (trimethylsilyl) trifluoroacetamide) (30 min at 60 ℃). After centrifugation (at 13300rpm, room temperature for 10 min) the derivatized samples were transferred to sample vials (sample injection within 24 hours). Finally, GC/MS detection analysis is performed on the treated sample.

GC-MS detection: GC-MS detection was performed on a gas chromatography system coupled to a mass spectrometer (Agilent 7890/MSD 5977B, calif., USA). The trimethylsilylated samples were separated using a DB-5MS capillary column (30 m 250 μm,0.25 μm film thickness; agilent J & W Scientific, folsom, calif., USA). Helium was used as carrier gas at a flow rate of 0.5ml/min. The sample inlet temperature was 250 ℃. The temperature of the GC column box was set to 60 ℃ for the first 1 minute, then increased to 325 ℃ at a rate of 10 ℃/min, and finally maintained at 325 ℃ for 10 minutes. The temperatures of the transmission line and the ion source were 300 ℃ and 230 ℃, respectively. The raw data of the mass spectrum were obtained using electron impact ionization (70 eV) in full scan mode (m/z 50-600). The residence time per scan was set to 1562u/s and the solvent delay was set to 5.9min. All samples were sampled in a random order with a sample volume of 0.5 μl.

Data preprocessing and analysis: statistical analysis was performed on the mass spectrum data using commercial software Agilent MassHunter and R software (version 3.4.0). The single-variable analysis and the multi-variable analysis are adopted to screen the differentially expressed metabolites (the screening conditions are (1) t-test, p value <0.1, and (2) OPLS-DA (orthogonal partial least squares discriminant analysis), VIP score > 1).

Data preprocessing: and (3) introducing mass spectrometry off-machine original data into commercial software analysis (version 1.6.3) and multi-quation (version 3.0.2) to perform peak extraction, and obtaining information such as mass-to-charge ratio, retention time, peak area and the like related to the metabolites. Mass spectrum data extracted by Progenesis QI software were then pre-processed with R software, including correction of sample mass bias, removal of low mass ions (more than 50% missing in QC samples, or more than 50% missing in real samples), removal of unstable ions (relative bias in all QC samples (RSD) >20% ion filtering out), missing value filling (KNN algorithm), eigems normalization (median), PCA showed distribution of Quality Control (QC) samples. The trend of the metabolites before and after seizure was analyzed by an unsupervised machine learning method FCM, and the metabolites were grouped (clustering) and the overall trend of the changes of each group was obtained.

Example III

In the present invention, a total of 93 urine samples were analyzed. The samples were divided into three groups according to the time of collection: n (normal state, i.e. the time of collection of the urine sample is 1 week before the seizure), B (pre-seizure, i.e. the time of collection of the urine sample is 1 to 3 days before the seizure), and a (post-seizure, i.e. the time of collection of the urine sample is within 1 hour after the seizure). The 77 metabolites in the urine samples were analyzed by FCM, which resulted in the final:

(1) There are 19 (as shown in the "male patient" column of fig. 10) metabolite levels that significantly increased 1-3 days prior to seizures in male patients (fig. 1, fig. 2, male cluster2 (cluster 2)) and significantly decreased after seizures. Specifically, figure 1 shows that FCM analysis forms the metabolites into three clusters (i.e., male clusters 1,2, and 3). While figure 2 further shows (with reference to the cluster global trend assistance schematic line) that the metabolites in male cluster1 gradually decrease overall from N to B to a, whereas the metabolites in male cluster 3 have no apparent trend overall from N to B, gradually increase overall from B to a, and only the metabolites in male cluster2 significantly increase overall from N to B, while significantly decrease overall from B to a.

(2) There are 16 (as shown in the "female patient" column of fig. 10) with significantly elevated levels of metabolites throughout 1-3 days prior to seizures in female patients (fig. 5, 6, female cluster1 (cluster 1)) and significantly reduced throughout after seizures. Specifically, figure 5 shows that FCM analysis forms the metabolites into three clusters (i.e., female clusters 1,2, and 3). While figure 6 further shows (with reference to the cluster global trend assistance schematic line) that the metabolites in female cluster2 gradually decrease from N to B to a, whereas the metabolites in female cluster 3 have no apparent trend throughout from N to B, gradually increase throughout from B to a, and only the metabolites in female cluster1 significantly increase throughout from N to B, while significantly decrease throughout from B to a.

Example IV

Further studies of the metabolite changes trend for the seizure cases of specific patients (8 actual seizure cases in the male and female groups) revealed that the levels of L-threonine (fig. 3) and homovanillic acid (fig. 4) were significantly elevated before seizures in male patients. And, even in the case of individual male episodes in which one of the above-mentioned metabolites is not elevated, the other metabolite is elevated (for example, L-threonine of male case episode number 7 is not elevated before episode (B) but elevated homovanillic acid is remarkable; homovanillic acid of male case episode number 5 is not elevated before episode (B), but elevated L-threonine is particularly remarkable).

In addition, the levels of L-threonine (fig. 7) and glycine (fig. 8) were significantly elevated prior to seizures in female patients. And, even in the case of individual female episodes, there was no elevation of one of the metabolites described above, the elevation of the other metabolite occurred (e.g., L-threonine for female case episode numbers 5, 6 was only slightly elevated before episode (B), but their glycine elevation was particularly pronounced).

Overall, the use of the two sets of metabolites as detection markers for patients of different sexes, respectively, can be more targeted and more effective in predicting seizure risk. In addition, since the two groups of metabolites contain L-threonine (which is obviously increased in most male patients and female patients) as a universal biomarker for predicting epileptic seizure, the number of detection markers can be reduced, and the cost of the detection scheme of the invention can be reduced.

This example further demonstrates the reliability of seizure prediction with a significantly elevated metabolite prior to seizure for both male and female patients. As shown in fig. 11 and 12, it was found that an elevated level of either threonine or homovanillic acid or glycine alone does not necessarily allow a better assessment of the risk of seizures, since there is a risk of "false negatives" (i.e. no elevation occurs before a seizure). However, the risk of seizures can be evaluated preferably by combining L-threonine with homovanillic acid (male patient) or L-threonine with glycine (female patient) (hereinafter referred to as a marker group), because the occurrence of elevation of both metabolites in the marker group is not present (0% in FIG. 11-L-threonine and homovanillic acid before seizures in male patient; 0% in FIG. 12-L-threonine and glycine before seizures in female patient), and the highest occurrence of elevation of both metabolites in the marker group (60% in FIG. 11-L-threonine and homovanillic acid before seizures in male patient; 43.75% in FIG. 12-L-threonine and glycine before seizures in female patient).

Based on the effect of the two sets of markers in predicting seizures, in some embodiments of the invention, when the content of all markers in a subject's marker set is not increased (same as below compared to the reference content), then the subject is assessed as seizure negative, i.e., the subject has a lower probability of seizures (can be considered as not seizures without taking precautions or interventions in advance) within 1-3 days of the biological sample being taken. When the content of one or both markers in a subject's marker set is increased, then the subject is assessed as seizure positive, i.e. the subject has a higher probability of seizures within 1-3 days of the biological sample being taken, and a preventive or interventional measure should be recommended to be taken by the epileptic patient in advance. When the content of all the markers in the marker group of the subject is increased, the subject is evaluated as positive for epileptic seizure, preventive measures or intervention measures are clearly recommended to be taken in advance by epileptic patients, and the possible harm caused by epileptic seizure is reduced (as shown in fig. 13).

The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are to be protected by the present invention.

Claims (6)

1. Use of a metabolic marker set comprising L-threonine, homovanillic acid and glycine in the manufacture of a kit for assessing the risk of seizures in a subject diagnosed with seizures and having a predisposition to seizures, said kit comprising a detection reagent for detecting the content of said metabolic marker set in a urine sample of said subject, said subject being male or female;

the L-threonine and homovanillic acid are used to assess the seizure risk in a male subject when the subject is male, the male subject being assessed as strongly positive for seizures when the content of both L-threonine and Gao Xiangcao acid is higher than a reference content to which it is referenced; when the content of the L-threonine or the homovanillic acid is higher than the reference content to which it refers, the male subject is assessed as seizure positive; when neither the content of the L-threonine nor the homovanillic acid is higher than the reference content to which it is referenced, the male subject is assessed as seizure-negative;

when the subject is female, the L-threonine and the glycine are used to assess the seizure risk in a female subject; when the content of both the L-threonine and the glycine is higher than the reference content to which it is referenced, the female subject is assessed as strongly positive for seizures; when the content of the L-threonine or the glycine is higher than the reference content to which it refers, the female subject is assessed as seizure positive; the female subject is assessed as seizure-negative when neither the content of the L-threonine nor the glycine is higher than the reference content to which it is referenced.

2. The use of claim 1, wherein the subject is at least 18 years old.

3. The use of claim 1, wherein when the level is higher than a reference level to which it is referenced, the subject is assessed as having a seizure risk for a predicted period of 1-3 days from the day of collection of the urine sample.

4. The use of claim 1, wherein the subject is a drug refractory epileptic patient.

5. The use of claim 1, wherein the subject has seizures at least once a month.

6. The use according to claim 1, wherein the detection is performed by one or more selected from chromatography, mass spectrometry, a combination of chromatography and mass spectrometry, immunochemistry, fluorescence and radiochemical.

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